Various methods for the total determination of hormones or pharmaceuticals present in human serum, bonded partially to specific or non-specific bonding proteins, are known. In recent years, the relatively new radioimmunological techniques have contributed substantially to the exact determination of hormones.
Detailed descriptions of radioimmunoassays are found, for example, in Clinical Chemistry, Vol. 19, No. 2, 1973, p. 145. In Clinical Chemistry, Vol. 19, No. 12, 1973, p. 1339, and Clinical Chemistry, Vol. 21, No. 7, 1975, p. 829, radioimmunological techniques are described in which an antibody enclosed in a gel is used. The inclusion of the antibody in gel is considered advantageous by the authors, because in this manner interactions with molecules of high molecular weight are excluded. However, these known working processes are not intended for total hormone determination. Nevertheless, from a medical standpoint, total hormone determination is essential.
Methods of the total determination of hormones through the enzymatic hydrolysis of the bonding proteins and with radioimmunological techniques are known from J. Clin. Endocrinol. Metab. 42, 189, 1976 and Clinical Chemistry, Vol 22, No. 11, 1976, p. 1850. These articles show that enzymatic cleavage has significant advantages compared with methods used heretofore, such as, for example, solvent extraction and heat denaturation. Advantages specifically named are the facts that extraction with organic solvents and the chromatographic purification of samples are eliminated, that the time and expense of the determination is reduced, that scinitallation counting in the liquid is eliminated, that specificity and accuracy is improved, that the method of determination can be automated, and that the method is universally applicable.
However, the enzyme used to hydrolyze the bonding protein also attacks the antibody introduced in the next working step and destroys the latter. For this reason, the enzyme must be deactivated prior to the addition of said antibody. Inactivation of enzymatic activity is possible in a number of ways, for example, the enzyme can be denaturated by a substantial change in the pH value or by a heat effect.
However, this denaturization step is extremely disadvantageous. It requires either the addition of supplemental chemicals which introduces still more factors of interference in the system or it requires long periods of time for heat denaturization. The heat effect must last for approximately 5 minutes, while the cooling period amounts to approximately 15 minutes. This denaturization step thus represents a substantial hindrance in the automation of the determination. Further, depending on the system applied, specifically the enzyme used, different types of denaturization must be performed, which opposes the general applicability of the method of determination and its automation.